INTRODUCTION:

Dental and oral disease commonly experienced by the societies in Indonesia is dental caries. In general, dental and periodontal caries is caused by dental plaque. Dental plaque is a sticky film populated by bacteria and formed products coating the dental surface¹.

According to² Indonesian citizens suffering dental and oral problem are found to be 25.9%. Dental caries can be prevented by using antibacterial agents (precipitating bacterial protein)^3,4. Quercetin, polyphenolics, quinone, saponins, alkaloids, and flavonoids can inhibit the bacterial growth. Furthermore, the antibacterial activities are also observed in guayaverin, leucocyanidin, essential oils, malic acid, damar and oxalic acid. The use of bioresources is taken as an option owing to its bacterial inhibitory properties (bacteriostatic) or its ability in killing the bacteria (bactericide)^5,6.

S. Mutans is a gram-positive bacterium which metabolizes carbohydrates, especially sucrose, contributing to the increase of the acidity in oral cavity^7,8. This condition leads to the formation of dental caries (Lamont, 2014). Guava leaves extract (Psidium guajava Linn.) was reported by⁵ to be capable of inhibiting S. Mutans at a concentration of 30%. Meanwhile, the maximum concentration of guava leaves extract, which is effective in inhibiting S. Mutans, is 100%. Higher extraction was reported to give higher inhibitory of S. Mutans^9,10.

METHODOLOGY:

Research Design:

This study is experimental research using a laboratory setting with post-test group design.

Used Model:

The model used in this research is the steep water of guava leaves which were collected from Sare, Aceh, Seulawah Agam Mountain. As for the S. Mutans, it is obtained from the Microbiology Laboratory of Faculty of Veterinary Medicine, Universitas Syiah Kuala.

Observed/Measured Parameters:

a. Changes of color were observed in phytochemical investigation.

b. MIC, the change of effectiveness was determined by observing the turbidity. High turbidity suggested the absence of bacterial inhibitory, while low turbidity was the otherwise. The presence of living bacteria was denoted with (+), and (-) for the otherwise.

Research Location:

The research was conducted at the Research Laboratory of Faculty of Mathematics and Natural Sciences, and Microbiology Laboratory of Faculty of Veterinary Medicine, Universitas Syiah Kuala, Banda Aceh, in 2019.

Number of Sample:

Fresh guava leaves samples were collected as many as 1 kg with a determination that of 1 kg fresh sample would have a weight reduction up to 400 g dried samples. In 1 kg fresh sample, the samples were sorted, where leaf samples which exhibited bad quality were excluded. The exclusion was made if the sample was found to have bad color, yellowish color, degraded leaves, fungus, caterpillar bite mark, and immaturity.

Preparation of Guava Leaves Steep Water:

1. Preparation of ‘tea powder’ used this following equation: 100% = 100 g/100 ml water

2. The water was boiled in 100 ml water until the temperature reached 100 °C.

3. Afterward, it was left cold until the temperature fall to 70-80 °C. The guava leaves simplisia/‘tea powder’ (100% 1 g/ml) was added¹¹.

4. It was covered and left cold until 30 °C, filtered and investigated for the MIC.

Determination of MIC Using Microdilution

1. Microplate with 96 wells (8 rows and 12 column was prepared¹².

2. Well 1,2,3,4,5,6,7,8 of the first row were filled with 150 µļ BHI (Brain Heart Infusion) liquid as the medium.

3. Then, the guava leaves steep water with a concentration of 100% (1 g/ml), as much as 150 µļ was added to well 1,2,3,4,5,6,7,8 of the first row, respectively.

4. Serial dilution was then carried out, where it gave concentrations of 100%, 50%, 25%, 12.5%, 6.25%, 3.125%, 1.563%, 0.753%. (Mardiastuti, et al., 2004).

5. Next, into all wells (1,2,3,4,5,6,7,8), S. Mutans was added as much as 10 μl¹².

6. After that the microplate was incubated in a facultatively anaerobic desiccator at 37 °C for 48 h.

7. The MIC was determined based on the turbidity of the culture and the reading of incubation result using MIC is the minimum concentration that gives a negative value¹².

8. It was then inserted into an indicator at 37 °C for 48 h, where the bacterial colonies can be observable, indicating the growth of the bacteria in MHA. It was then compared with the sterile MHA, where the colonies were not found. MIC was determined to express the minimum concentration where the growth of S. Mutans was not observed¹².

9. The test was conducted for three times repetition.

RESULTS AND DISCUSSION:

Guava leaves as many as 1 kg were collected as samples, washed clean and left dried at room temperature for 14 days. The dried leave samples were put in a crusher to yield a simplisia (Biswas et al., 2011). The simplisia was investigated by phytochemical test.

Results of Phytochemistry of Guava Leaves Steep Water:

The phytochemical test aimed to identify the secondary metabolites in a plant. Phytochemical test includes alkaloids, steroids, terpenoids, saponins, flavonoids, and phenolics test. Alkaloids test can be conducted by adding mayer, dragendorff, and wagner reagents, respectively. The addition of mayer reagent formed white precipitates suggesting the presence of alkaloids. Meanwhile the addition of dragendorff showed red precipitates and wagner showed brown precipitates for alkaloids-positive samples. Phytochemical test of steroids and terpenoids can be conducted by using Liebermann-Burchard reagent; if the color turned into red, the sample is positive containing terpenoids, whilst if the color turned into green or blue it is positive containing steroid. Phytochemical test of flavonoids can be done by extracting the residue with ethanol and adding HCl and Mg powder to yield a pink or purple color, showing the presence of flavonoids^14-16.

The results of phytochemical test of fresh guava leaves reveal the secondary metabolite contents of terpenoids, steroids, saponins, flavonoids and phenolics. The presence of terpenoids, along with the steroids are very common to be found in plant owing to the biosynthesis pathway of steroid being originated from terpenoids

Minimum Inhibitory Concentration (MIC):

The determination of minimum inhibitory concentration (MIC) was carried out against the Streptococcus mutans. Microdilution was selected as the method to determine the MIC. The value is an expression of a minimum concentration that could give the first negative mark.

Table1. Minimum inhibitory concentration

S. No	Concentration (%)	MIC
		I	II	III
1.	100	-	-	-
2.	50	-	-	-
3.	25	-	-	-
4.	12,5	-	-	-
5.	6,25	-	-	-
6.	3,125	+	+	+
7.	1,563	+	+	+
8.	0.781	+	+	+
9.	Aqua	+	+	+

Information = the growth is present (+), the growth is absent (-)

Table 1 shows that the guava leaves steep water at a concentration of 6.25% gave the first negative value. This value indicated the inhibition of the bacteria. Therefore, the MIC of guava leaves steep water is 6.25%.

DISCUSSION:

Phytochemical test on the guava leaves steep water includes the test of alkaloids, steroids, terpenoids, saponins, flavonoids, and phenolics. Alkaloids test can be done by adding the mayer, dragendorff, and wagner reagents. The addition of mayer reagent formed white precipitates suggesting a positive presence of alkaloid contents. Meanwhile, dragendorff reagent gave red precipitates and wagner reagent gave a brown precipitate, confirming the presence of alkaloids in the sample. Phytochemical test of steroids and terpenoids could be done using Liebermann-Burchard reagent, where the color changing into red indicated the presence of terpenoids. Meanwhile, the changing into green or blue color suggested the presence of steroid. Phytochemical test of flavonoids was carried out by extracting the residue with ethanol, followed by the addition of HCl and Mg powder to yield a pink or purple color, which suggested the presence of flavonoids.

From the phytochemical test on guava leaves steep water, the observed secondary metabolites included terpenoids, steroids, saponins, flavonoids, and phenolics. The presence of terpenoid alongside with steroids is very general in plant. It is owing to the fact the that the steroids are biosynthesized from terpenoids

The results of antibacterial activities test of guava leaves steep water against S. Mutans show that the guava leaves steep water had antibacterial activities. The MIC of the guava leaves steep water, which is the minimum concentration required to inhibit the bacteria (giving negative values), was 6.25%. The inhibition of S. Mutans growth was suspected to be associated with the presence of the bioactive compounds in the guava leaves steep water; such as alkaloids, flavonoids, tannins, saponins, polyphenols which contributed to the bacterial mortality.

Alkaloids have the ability as an antibacterial by interfering the constituent components of the cellular peptidoglycan of the bacteria, leading to defective formation of the layers of the cell wall. This interreference eventually lead to the cell death. Alkaloids, phenol, was an antitoxic which caused the three-dimensional protein of the bacteria to be interfered and converted into a random structure, causing a protein denaturation. This further led to the damage on the biological activities, so that the growth of S. Mutans was stopped. (Khairi, 2016).

Furthermore, flavonoids have a function as an antibacterial by forming a complex compound with the extracellular protein causing a damage on the structure and the change in cellular membrane mechanism. Flavonoids (phenol) inhibited the synthesis of the bacterial nucleic acids, as well as the motility of the bacteria¹⁸.

Guava leaves steep water also contains saponins which can act as a strong surfactant agent (like a soap). It is due to the fact it has the ability to reduce the tension of the intercellular tension. Saponins were absorbed on the cellular surface causing a damage by increasing the membrane permeability, so that the bacteria experienced a loss in the essential nutrients, causing a cell death (Khairi, 2016). Saponin can inhibit the bacterial growth which is attributed to its ability in reducing the tension of the intercellular surface of the bacterial cell wall. When the interaction occurs on the bacterial cell wall, it causes a breakdown or lysis. It is then followed by the entry of the antibacterial agents into the bacterial cell, leading to the interference of the bacterial metabolism and killing the bacteria¹⁷.

Tannins contained in the guava leaves steep water can cause a shrinkage of the cell wall or cell membrane, interfering the cellular permeability. Hence, it can lead to a cell death. Polyphenol has a potential as an antibacterial by toxicating the protoplasm and damaging the bacterial cells²⁰.

CONCLUSION:

1. Guava leaves steep water was effective in inhibiting the Streptococcus mutans bacteria.

2. The minimum inhibitory concentration (MIC) was found to be 6.25%.

RECOMMENDATION:

1. Further studies on guava leaves steep water as an antibacterial mouthwash against the Streptococcus mutans have to be conducted.

2. The next research will take the guava leaves as an ingredient for tooth paste preparation.

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Received on 13.07.2020 Modified on 08.02.2021

Research J. Pharm. and Tech 2021; 14(11):5745-5748.

DOI: 10.52711/0974-360X.2021.00999